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P Rovero

Publications and source records attributed to P Rovero.

At least 37 records · Page 2Linked to original sources
PubMed1997

Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.

A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1(22-38), the C-terminal of ET-1, six analogues of ET-1(16-21) each containing a substitution with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, with less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38). Ala substitution in positions 16, 17 and 19 of ET-1(16-21) hardly affected the antibody binding capacity of ET-1(16-21), whereas Ala substitution of Asp18, Ile20 and, in particular, Trp21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(15-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.

Amino Acid Sequence↗
PubMed1997

SPPS of difficult sequences. A comparison of chemical conditions, synthetic strategies and on-line monitoring.

The H-Ala-Arg-(Ala)6-Lys-OH sequence is a biologically interesting 'difficult sequence' presenting N alpha-Fmoc deprotection and coupling problems. Different chemical conditions and synthetic strategies have been tested in order to overcome the problems due to sequence-dependent interactions. In particular, it was confirmed that different solvents in the deprotection step did not provide any significant improvement, but the use of a more efficient base in the deprotection mixture avoided insufficient unblocking of N alpha-protecting group; problems due to partial coupling in the last steps of the synthesis were solved by double coupling techniques. Moreover, the synthesis of the model peptide was carried out using both "continuous flow' and "batch' techniques. The present results demonstrate that on-line monitoring of the deprotection step by absorbance measurements represents a very effective tool to detect the onset of internal aggregations during the synthesis.

Chromatography, High Pressure Liquid↗
PubMed1996

Inhibition of feline immunodeficiency virus infection in vitro by envelope glycoprotein synthetic peptides.

Sixty-six 20- to 23-amino-acid synthetic peptides, partially overlapping by 10-12 amino acids, spanning the entire sequence of the envelope SU and TM glycoproteins of the Petaluma isolate of FIV, have been used to investigate the Env domains involved in viral infection. Peptides 5 to 7, spanning amino acids 225E-P264 located in a conserved region of the SU protein, and peptides 58 to 61, spanning amino acids 767N-P806 and encompassing hypervariable region 8 of TM protein, exhibited a remarkable and specific antiviral effect against the homologous and one heterologous isolate, as judged by inhibition of FIV-induced syncytium formation and p25 production in CrFK cells. Peptides 5 and 7, but not peptides 58 and 59, also inhibited viral replication of a fresh FIV isolate on nontransformed lymphoid cells. By flow cytometry, peptides 5, 7, 58, and 59 were shown to bind the surface of FIV permissive cells. The antiviral activity of peptides 5 and 7, however, was time-dependent, as inhibition of FIV replication was seen when the peptides were administered before or within 3 hr after virus inoculation; in contrast, TM peptides 58 and 59 exerted a potent inhibitory effect when added up to 24 hr after virus inoculation. Circular dychroism analysis showed that peptide 5 folds to a helical conformation in the presence of a hydrophobic environment. Although the basis for the antiviral action of the peptides is not understood, our data suggest that the inhibitory peptides may act by interacting with cell-surface molecules involved in viral infection.

3T3 Cells↗
PubMed1996

Investigation of newly synthesized peptides by capillary zone electrophoresis/electrospray mass spectrometry.

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Amino Acid Sequence↗
PubMed1996

Putative odorant-binding protein in antennae and legs of Carausius morosus (Insecta, Phasmatodea).

A 19 kDa protein has been purified by gel filtration and anion-exchange chromatography from the antennae of Carausius morosus. Its amino terminal amino acid sequence shows significant similarity (30% identity) with another putative odorant-binding protein, the so called OS-D protein isolated from the antennae of Drosophila melanogaster; only 20% of its amino acids are shared with some members of Lepidoptera pheromone-binding proteins. Polyclonal antibodies, raised against a synthetic amino terminal peptide cross-react with 19 kDa band in the legs extracts, but not with soluble proteins from other parts of the body. The amino terminal sequence of this protein, purified from the legs was identical with that of the antennal protein.

Amino Acid Sequence↗
PubMed1996

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Antibodies, Monoclonal↗
PubMed1995

Autoantibodies directed against ribosomal P proteins: use of a multiple antigen peptide as the coating agent in ELISA.

Autoantibodies directed against the ribosomal proteins P0, P1 and P2 (P proteins) are specific for systemic lupus erythematosus (SLE) and there are some evidences that they could be related to the neuropsychiatric manifestations of the disease. In this study, a multiple antigen peptide (MAP) carrying four copies of the C-terminal peptide (13 residues) of the P2 protein, which is a common epitope of the three P proteins, was prepared for use in an ELISA assay. It was employed to detect antibodies directed against the ribosomal P proteins in 102 SLE patients and the results were compared with those obtained using immunoblotting (IB). With this new ELISA, antiribosomal P protein antibodies were found in 15/102 SLE sera. These results correlated well with the results of IB. Furthermore, we confirmed that naturally occurring antiribosomal P protein antibodies are directed mainly against the epitope containing the C-terminal sequence and shared by the three P proteins. MAP appears to be an excellent coating agent for ELISA assays designed to detect anti-P antibodies. Further experiments showed the superiority of MAP, compared to the free peptide, in the detection of weakly positive sera. This ELISA can also be used for the serological follow-up of SLE patients.

Amino Acid Sequence↗
PubMed1995

CD and fluorescence studies of the human granulocyte-macrophage colony-stimulating factor and related peptide conformations in aqueous solution.

The absorption, CD, and fluorescence emission spectra, and the fluorescence emission and depolarization lifetimes of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) and related peptides previously tested for their immunological activity, were measured in water at various pHs and temperatures to obtain information on their conformation in solution. The aim was to correlate the amino acid sequences, and the chain conformations and dynamics of the peptides, with their immunological properties. The CD spectrum of hGM-CSF revealed, as expected, a structure in solution similar to that in the crystalline state, but the fluorescence data suggest that the Trp 122 residue is more accessible to the solvent than the x-ray data would lead one to expect. They also suggest that some flexibility exists between the protein's two domains, one made up of the alpha-helices A and C and the other of the alpha-helices B and D plus the two beta-strands. In aqueous solution, none of the tested peptide CD spectra could be linked to a recognizable ordered conformation, i.e., an alpha-helix or a beta-sheet. The fluorescence of the peptide 11-24 suggests that the Trp 13 residue may appear in two types of situations: (a) in aqueous solution and (b) within a globular structure. Its CD spectra show that the tryptophan residue exists in both cases in a highly asymmetric environment independent of the pH.

Circular Dichroism↗
PubMed1995

Differences in peptide-binding specificity of two ankylosing spondylitis-associated HLA-B27 subtypes.

Two HLA-B27 subtypes, B*2702 and B*2705, both associated with ankylosing spondylitis, were tested for binding affinity with a panel of polyalanine model nonapeptides carrying Arg at position 2 (P2) and a series of different amino acids at position 9 (P9). The alpha chains were isolated from BTB(B*2705), C1R/B*2702 (a B*2702 transfectant cell line) and from the NW (B*2702) cell line that has a peculiar peptide presentation behavior. Peptide binding was measured by the HLA alpha chain refolding assay. The results obtained show that: 1) Peptides with basic residues (Arg and Lys) and also aliphatic (Leu) and aromatic (Phe and Tyr) peptides at P9 have a similar high affinity in the binding to B*2705; 2) B*2702 binds well to P9 aliphatic and aromatic peptides but only very weakly to P9 basic peptides. Since both B*2702 and B*2705 are associated with AS the presumed arthritogenic peptide is hypothesized to have an aromatic or aliphatic residue at position 9. Peptides with basic residues in this position would be excluded as candidates because of their low binding affinity with B*2702.

Amino Acid Sequence↗
PubMed1995

GR 73,632 and [Glu(OBzl)11]substance P are selective agonists for the septide-sensitive tachykinin NK1 receptor in the rat urinary bladder.

The existence of a septide-sensitive subtype of the tachykinin NK1 receptor has been recently proposed. In the rat isolated urinary bladder, the non-peptide NK1 receptor antagonist RP 67,580 exhibits a higher affinity towards septide (pKB 7.57) than towards [Sar9]substance P sulfone (pKB 7.00). In this study we have investigated the pharmacological profile of the non-mammalian tachykinin physalaemin, of the synthetic NK1 receptor agonist GR 73,632 (delta-aminovaleryl[LPro9,NMeLeu10]substance P(7-11)) and of [Glu(OBzl)11]substance P in relation to the putative existence of a septide-sensitive receptor. The activity of [Glu(OBzl)11]substance P at the NK1, NK2 and NK3 receptor was assayed in the guinea-pig ileum NK1 receptor assay (EC50 26 nM), in the rabbit pulmonary artery NK2 receptor assay (weak agonist activity) and in the rat portal vein NK3 receptor assays (no appreciable activity up to 1 microM). GR 73,632, [Glu(OBzl)11]substance P and physalaemin, all produced concentration-dependent contractions of the rat isolated urinary bladder, with EC50 values of 17, 79, and 9 nM, respectively. The responses to the three agonists were very slightly or not modified by the NK2 receptor antagonist SR 48,968 (1 microM). RP 67,580 (0.3-3 microM) produced a concentration-dependent rightward shift of the curve to GR 73,632, [Glu(OBzl)11]substance P and physalaemin without producing depression of their maximal response. Schild plot analysis indicated the competitive nature of the antagonism. The affinity (pKB) of RP 67,580 towards physalaemin, GR 73,632 and [Glu(OBzl)11]substance P was 7.12, 7.56 and 7.95, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A new endothelin C-terminal analogue IBDP 064 antagonizes endothelin-3-induced cell proliferation.

A series of C-terminal linear endothelin analogues were prepared and their activities in C6 rat glioma cell line were tested. Among the synthetic analogues, IBDP 064, Fmoc-Leu-Asp-Ile-Ile-Trp-OH, was the most potent and selective inhibitor of endothelin-3-induced cell proliferation. Its action was comparable with that of the previously described peptide IRL 1038, [Cys11-Cys15]-ET-1(11-21), an ETB specific inhibitor.

Amino Acid Sequence↗
PubMed1994

The peptide binding specificity of HLA-B27 subtypes.

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.

Amino Acid Sequence↗
PubMed1994

Synthesis and biological activity of NK1 substance P selective agonists by modifying the methionyl residue.

A series of analogues of substance P, where the Met11 residue was replaced by Glu(OCH2CH3), Glu(OBzl) and Hse(CH3), were synthesized in order to investigate the effect on agonist activity of modifications at the C-terminal residue. The biological activities in the guinea-pig ileum assay (NK1 receptor) and rat [text says rabbit] pulmonary artery (NK2 receptor) indicate that replacement of the SCH3 group of Met11 of substance P by the COOBzl or OCH3 groups favour interaction with the NK1 receptor and increase selectivity towards this receptor subtype.

Amino Acid Sequence↗
PubMed1994

Synthesis and biological activity of NK1 tachykinin antagonists not containing D-residues.

A series of analogues of the previously reported NK1 tachykinin antagonist [Orn6, Glu(OBzl)11]-SP6-11-OBzl has been synthesized and tested in order to investigate the effects on antagonistic activity of modifications at the C-terminal residue. The biological activity in the guinea-pig ileum assay (NK1 receptor) indicates that the two aromatic rings introduced in the C-terminal part of the peptide are both essential for the expression of antagonistic activity.

Amino Acid Sequence↗
PubMed1994

The importance of secondary anchor residue motifs of HLA class I proteins: a chemometric approach.

In this paper we report a chemometric approach to Quantitative Structure-Activity Relationship (QSAR) analysis applied to a study of the binding of peptides to Major Histocompatibility Complex (MHC) class I proteins. Peptides which possess the known primary anchor residue motif for HLA-B27 binding do not necessarily bind to HLA-B27 proteins. Secondary anchor residues are also involved, but it is not yet clear which amino acids are required or in which positions. A classic approach to this problem would be to synthesize multiple peptides each varying by a single amino acid from a starting peptide, and test them for their binding properties. Not only is this approach inefficient, but it is essentially unable to provide information about possible mutual interactions of amino acid residues in different positions. Using a statistical design to select the most informative compounds to use in the QSAR study, it was possible to analyse the effects on HLA-B27 peptide binding of different amino acids in four positions by means of only nine peptides. The relative binding activity of these peptides could then be modeled mathematically to provide information about the relative contribution of each of the four positions and to suggest a new peptide with high binding affinity. Our results demonstrate the usefulness of the chemometric strategy for studying peptides of interest in molecular immunology.

Amino Acid Sequence↗
PubMed1994

An immunodominant epitope in a functional domain near the N-terminus of human granulocyte-macrophage colony-stimulating factor identified by cross-reaction of synthetic peptides with neutralizing anti-protein and anti-peptide antibodies.

We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

Amino Acid Sequence↗
PubMed1994

Solution conformation of c-[Gln-Trp-Phe-Gly-Leu-Met], a NK-2 tachykinin antagonist.

The conformation of cyclo-[Gln-Trp-Phe-Gly-Leu-Met], a potent tachykinin antagonist selective for the NK-2 receptor, has been studied by 1H NMR spectroscopy in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture in the temperature range 280-320 K. The NMR data cannot be interpreted on the basis of a single ordered conformation. An exhaustive search, based mainly on missing NOEs among skeleton protons, yields a description of the conformational state in solution consisting of a few interconverting structures that can explain all observed NMR parameters. The relative position of the side chains of key residues may be interpreted in terms of bioactive conformations.

Amino Acid Sequence↗